1. Field of the Invention
This invention relates to the fields of nucleic acid constructs and cell lines that allow for the increased expression of endogenous or heterologous target protein.
2. Background
The immediate challenge created by the genomics era is the production of the novel proteins to understand their function. Current methods of expressing genes in a mammalian cell include the use of viral vectors, such as those which are derived from retroviruses, adenoviruses, herpes viruses, vaccinia viruses, polio viruses, sindbis viruses, or adeno-associated viruses. Other methods of expressing an exogenous gene in a mammalian cell include direct injection of DNA, the use of ligand-DNA conjugates, the use of adenovirus-ligand-DNA conjugates, calcium phosphate precipitation, and methods which utilize a liposome- or polycation-DNA complex.
Due to its advantages in versatility and speed, the Baculovirus Expression Vector System (BEVS) used in conjunction with insect cells has become well-established for the production of proteins, particularly recombinant glycoproteins. Baculovirus mediated protein expression provides correct folding of recombinant protein as well as disulfide bond formation, oligomerization and other important post-translational modifications that impart proper biological activity and function. With regard to protein folding and post-translational processing, insect cells are second only to mammalian cell lines when expressing a eukaryotic protein, for example. The frequent use of baculovirus arises from the relative ease and speed with which a heterologous protein can be expressed on the laboratory scale and the high chance of obtaining a biologically active protein. Insect cells can be grown on serum free media which is an advantage in terms of costs as well as of biosafety. For large scale culture, conditions have been developed which meet the special requirements of insect cells.
In nature, baculoviruses are double-stranded DNA-containing viruses that infect a variety of different insect species. The nuclear polyhedrosis viruses, which comprise subgroup A of the Family Baculoviridae, induce the formation of paracrystalline occlusion bodies in the nuclei of infected host cells. These occlusion bodies are composed primarily of a single viral protein which is expressed at very high levels (polyhedrin). In later stages of the infection cycle, polyhedrin may account for more than 50% of the total protein in an infected cell. The polyhedrin gene has been cloned and sequenced and its unique features have provided the basis for the development of a series of baculovirus expression vectors (BEVs: Summers, M. D. and Smith, G. E., TAES Bull. 1555 (1987); Luckow, V. A. and Summers, M. D., Biotechnology 6:47-55 (1988); Miller, L. K., Ann. Rev. Microbiol. 42:177-179 (1988); U.S. Pat. No. 4,745,051, G. E. Smith and M. D. Summers (Filed May 27, 1983; Issued May 17, 1988)).
BEVs are recombinant baculoviruses in which the coding sequence for polyhedrin has been replaced with the coding sequence for a desired protein. In general, this approach involves the construction and isolation of recombinant baculoviruses in which the coding sequence for the chosen gene has been inserted behind the promoter for the nonessential polyhedrin viral gene (Pennica, et al, Mol. Cell. Biol. 4:399-406 (1984); Smith, et al, L. Virol. 46:584-593 (1983); Smith, G. E. and M. D. Summers, Mol. Cell. Biol. 3:2156-2165 (1983). Several advantages may exist when employing the BEV system. One of these advantages is the strong polyhedrin promoter which directs a high level of expression of the inserted heterologous nucleic acid encoding the target polypeptide. The newly expressed heterologous target protein accumulates in large amounts within these infected insect cells. Thus, as a result of the relative strength of the polyhedrin promoter, many different gene inserts can be expressed at very high levels.
In addition to providing a high expression level, another advantage of the BEV system is the ease with which these baculoviruses are produced and identified. This process begins by co-transfecting wild-type viral DNA and a “transfer vector” into susceptible host cells. A transfer vector is defined as a bacterial plasmid which contains a desired gene directly 3′ to the polyhedrin promoter, as well as long viral sequences flanking the promoter on the 5′ side. During cotransfection, homologous recombination occuring between viral and transfer vector DNA will produce a small percentage of viral genomes in which the polyhedrin gene has been replaced by the desired heterologous nucleic acid encoding the target polypeptide (0.1-5.0%). The wild-type progeny can be differentiated from the recombinant progeny by a conventional viral plaque assay. Recombinants in which the polyhedrin gene has been replaced, can be identified by their occlusion-negative plaque phenotype observed in a background of occlusion-positive wild-type plaques.
Because the polyhedrin gene is a non-essential gene for productive viral infection, another advantage of baculovirus expression vectors is that the recombinants are viable, helper-independent viruses. Also, baculoviruses only infect Lepidopteran insects; thus, they are noninfectious for vertebrates, and are, therefore, relatively safe genetic manipulation agents.
Notwithstanding the successes of BEVS and other systems for expression of heterologous proteins in insect and mammalian cell culture, maintenance of the viability of transformed or transfected cell cultures remains a capricious undertaking. Many laboratories refer to tissue culture as a “black art,” due to the numerous variables that make it difficult to determine solutions when problems arise. An intensive and time-consuming systematic approach that examines the symptoms and meticulously retraces each step in the culture process is usually required to identify the material or critical procedure that has created the viability issue. Problems such as poor cell growth and abnormal morphology can result from materials that are poor quality, inappropriate, compromised, or contaminated and/or equipment that must be re-calibrated or re-setup to comply with manufacturer usage. Perhaps most frustrating, cells of different lots may react differently to standardized media and serum supplements resulting in unexpected toxicity or nutritional deficiency. Therefore, much of the time and expense invested in preparation of protein expression vectors may be lost when a protein production facility experiences difficulty in optimizing cell culture protein production conditions. As such it would be of great economic benefit to provide a generalized agent to a cell line to increase its viability, longevity and protein production capacity.
Insects, like other animals, have effective immune systems to combat both biotic and abiotic foreign invasion. Interestingly, endoparasitic insects spend a part of their life cycle inside the body of other insect hosts. Considerable effort has been expended investigating the mechanism by which these endoparasitic insects avoid the host immune system in this parasitic relationship.
One well characterized parasitoid-host system in which there is immune system evasion is that of the endoparasitic wasp Campoletis sonorensis and its host, the tobacco budworm Heliothis virescens. In investigating how immunosuppression is regulated in this system, it became apparent that a group of wasp viruses, known generically as polydnaviruses (PDVs), play a role in the suppression of the host immune system. Bracoviruses (BVs) and ichnoviruses (IVs) are the two main parasitic wasp associated PDVs. It is known that during oviposition, the endoparasitic insect, for example C. sonorensis, injects not only eggs but also polydnavirus and oviduct proteins. Shortly thereafter, the host insect immune system begins to show evidence of altered activity and the endoparasitoid eggs remain free from encapsulation. The precise mechanism of this immune suppression is not presently understood.
The WHv1.0, WHv1.6 and VHv1.1 genes of C. sonorensis polydnavirus (CsPDV) have been cloned and sequenced. These genes are described as members of a polydnavirus “cysteine-rich” gene family. (Dib-Hajj et al., Proc. Natl. Acad. Sci. (USA) 90: 3765 (1993)). It has been conjectured that these genes may play a role in preventing the recognition of foreign objects and/or the normal response of components of the immune system. (Summers et al., Proc. Natl. Acad. Sci. (USA) 92: 29 (1995)). Indeed, the VHv 1.1 gene product of the C. sonorensis polydnavirus has been implicated in the inhibition of the cellular immune response. This 30 kDa protein is shown by indirect immunofluorescence to bind both granulocytes and plasmatocytes and is thought to inhibit encapsulation. (Li et al., J. Virol., 68: 7482 (1994)).
Recent PDV genome sequencing projects have revealed a novel family of closely related genes that exist in several genomes including, but not limited to, the C. sonorensis IV (CsIV) Hyposoter fugitivus IV (HfIV), Glypta fumiferana IV (GfIV), Microplitis demolitor BV (MdBV), Cotesia congregata BV (CcBV), Glyptapanteles indiensis BV (GiBV), and Toxoneuron nigriceps BV (TnBV) genomes. This family of genes has been named vankyrins as their open reading frames (ORFs) encode proteins almost exclusively made up of ankyrin repeat domains. The PDV ankyrin repeat-carrying proteins show significant identity to the ankyrin repeats of the Iκβ family of transcription factor inhibitors suggesting that they disrupt intracellular NF-κβ mediated signal transduction cascades known to play a role in both vertebrate and invertebrate immune responses. There are seven vankyrin ORFs encoded by the CsIV genome.
The inventors have discovered that the expression of two CsIV vankyrins from a heterologous expression vector system increases the vitality, longevity, and therefore the protein productive capacity of cells in culture.
All references cited herein are hereby incorporated by reference in their entirety for all purposes.